Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H(2) (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol(-1)) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H(2) consumption by the methanogens, which lowers the H(2) partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H(2). Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H(2)-producing growth. The actual ΔG values for the formate metabolism are calculated to range between -8 and -20 kJ mol(-1) under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability.

Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO 2 , thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.Deep-sea hydrothermal vents comprise a plethora of potential habitats, with gradients of nutrient and extreme physicochemical conditions that vary from high to low with respect to temperature (350 to 2°C), oxygenation states, and fluid velocities (13). Many multidisciplinary studies have been carried out to understand the complexities of hydrothermal vent systems. Biological studies have also been accomplished using samples collected from hydrothermal vent areas and culture-dependent and culture-independent techniques, revealing the presence of physiologically, metabolically, and phylogenetically diverse microorganisms (15). These findings have been followed by characterization of many bacterial and archaeal thermophiles (and hyperthermophiles), including both chemolithoautotrophic and chemoorganoheterotrophic strains. Among representative species of the Archaea, sulfur-reducing heterotrophs belonging to the order Thermococcales (encompassing the genera Thermococcus, Pyrococcus, and Palaeococcus) have been reported to be one of the predominant groups (20, 25). Notably, members of the species of Thermococcus were found to be more abundant in the vent ecosystem, with such isolates more frequently reported than the Pyrococcus species (9,11,23,24). Such large populations indicate some significance for the presence of Thermococcus in the microbial consortia that make up the microbial ecology of hydrothermal vent systems.In addition to ecological significance, the hyperthermophilic feature of Thermococcales has fascinated microbiologists interested in fundamental and/or application-based research. To date, the complete genome sequences of three Pyrococcus species, i.e., Pyrococcus horikoshii (16), Pyrococcus furiosus (26), and Pyrococcus abyssi (5), and a Thermococcus strain, Thermococcus kodakaraensis KOD1 (7), have been determined. Analysis of the sequences and the physiolo...

bHydrogenogenic CO oxidation (CO ؉ H 2 O ¡ CO 2 ؉ H 2 ) has the potential for H 2 production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H 2 , has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H 2 , the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na ؉ /H ؉ antiporter and a 1.8-fold-higher specific activity for CO-dependent H 2 production than did the wild-type strain. The H 2 production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H 2 production rate of the engineered strain was severalfold higher than those of any other COdependent H 2 -producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H 2 production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe. Carbon monoxide (CO) is highly toxic to most living creatures, but it can be utilized by microorganisms as an energy and carbon source for the production of fuels and chemicals, such as acetate, butyrate, ethanol, butanol, and H 2 . Among those carboxydotrophic microbes, CO-dependent H 2 production has been observed in three distinct groups, i.e., mesophilic bacteria, thermophilic bacteria, and hyperthermophilic archaea (1-3). Generally, growth rates of the mesophilic hydrogenogenic bacteria on CO are low, and high levels of CO are inhibitory. Predominant within this group are nonsulfur purple bacteria, including Rubrivivax gelatinosus and Rhodospirillum rubrum, which require light for optimal cell growth. Although Rhodopseudomonas palustris P4 is capable of hydrogenogenic CO conversion in the dark, it does not grow under this condition (4). Nonphototrophic Citrobacter strain Y19 also converts CO to H 2 , but it only grows slowly under anaerobic conditions and an aerobic growth phase is required to generate sufficient biomass before the anaerobic CO conversion phase (5). The second group includes thermophilic, hydrogenogenic bacteria isolated from freshwater and marine environments with temperatures ranging from 40 to 85°C. Carboxydothermus hydrogenoformans, Carboxydocella thermautotrophica, Thermosinus carboxydivorans, and Caldanaerobacter subterraneus subsp. pacificus are capable of chemolithotrophic growth on high concentrations of CO. The third group includes two hydrogenogenic CO-converting archaea, Thermococcus sp. strain AM4 and Thermococcus onnurineus NA1 (6, 7). Both strains are hyperthermophiles isolated from deep-sea hydrothermal vents and can grow on 100% CO (8).Anaerobic carboxydotroph...

The hyperthermophilic archaeon, Thermococcus onnurineus, was grown in media supplemented with either CO, formate, or starch. H(2) was produced with each substrate with respective maximum rates of 1.55, 3.83 and 2.66 mmol H(2)/l h. The yields (mol H(2)/mol substrate) were 0.98, 1 and 3.13, respectively. This microbe is the first example where a single microorganism can grow and produce H(2) using CO, formate or starch as substrate.